1. Field of the Invention
This invention relates to genetic modification of fungi to enhance production of a protein of interest. Furthermore, this invention relates to novel genetic constructs that dramatically increase the amount of protein produced by fungi containing these constructs.
2. Background of the Related Art
The use of fungal expression systems for the production of proteins of interest is well known within the art. For example, heterologous proteins have been produced within fungal expression systems for biomass conversion, detergent applications, de-pilling of cellulase substrates and other industrial enzyme uses. The production of other heterologous proteins of interest, such as food additives or supplements, pharmaceutical compounds, antibodies, protein reagents and the like, and industrial proteins is also feasible within fungal expression systems.
Many microbes make enzymes that hydrolyze cellulose, including the wood rotting fungus Trichoderma, the compost bacteria Thermomonospora, Bacillus, and Cellulomonas; Streptomyces; and the fungi Humicola, Aspergillus and Fusarium. The enzymes made by these microbes are mixtures of proteins with three types of actions useful in the conversion of cellulose to glucose: endoglucanases (EG), cellobiohydrolases (CBH), and β-glucosidase. EG and CBH enzymes are collectively referred to as “cellulase”. EG enzymes cut the cellulose polymer at random locations, opening it up to attack by CBH enzymes. As an example, Trichoderma strains produce at least four distinct EG enzymes, known as EGI, EGII, EGIII, and EGV. CBH enzymes sequentially release molecules of cellobiose from the ends of the cellulose polymer. Cellobiose is the water-soluble β-1,4-linked dimer of glucose. There are two primary CBH enzymes within Trichoderma, CBHI and CBHII. β-glucosidase enzymes hydrolyze cellobiose to glucose. Trichoderma makes one β-glucosidase enzyme.
This final step in the cellulose hydrolysis which is catalyzed by β-glucosidase is important, because glucose is readily fermented to ethanol by a variety of yeasts while cellobiose is not. Any cellobiose remaining at the end of the hydrolysis represents a loss of yield of ethanol. More importantly, cellobiose is an extremely potent inhibitor of the CBH and EG enzymes. Cellobiose decreases the rate of hydrolysis of the Trichoderma CBH and EG enzymes by 50% at a concentration of only 3.3 g/L. The decrease in rate of hydrolysis necessitates the addition of higher levels of cellulase enzymes, which adversely impacts the overall process economics. Therefore, the accumulation of cellobiose during hydrolysis is extremely undesirable for ethanol production.
Cellobiose accumulation has been a major problem in enzymatic hydrolysis because Trichoderma and the other cellulase-producing microbes make very little β-glucosidase. Less than 1% of the total protein made by Trichoderma is β-glucosidase. The low amount of β-glucosidase results in a shortage of capacity to hydrolyze the cellobiose to glucose and an accumulation of 10 to 20 g/L of cellobiose during hydrolysis. This high level of cellobiose increases the amount of cellulase required by 10-fold over that if an adequate amount of β-glucosidase were present.
Several approaches have been proposed to overcome the shortage of β-glucosidase in cellulase enzymes.
One approach has been to produce β-glucosidase using microbes that produce little cellulose, and add this β-glucosidase exogenously to cellulase enzyme to enhance the hydrolysis. The most successful of such β-glucosidase producing microbes have been Aspergillus niger and Aspergillus phoenicis. B-glucosidase from these microbes are available commercially as Novozym 188 from Novo Nordisk. However, the quantities required are much too costly for a commercial biomass to ethanol operation.
A second approach to overcoming the shortage of β-glucosidase is to carry out cellulose hydrolysis simultaneously with fermentation of the glucose by yeast, the so-called simultaneous saccharification and fermentation (SSF) process. In an SSF system, the fermentation of the glucose removes it from solution. Glucose is a potent inhibitor of β-glucosidase, so SSF is an attempt to increase the efficiency of β-glucosidase. However, SSF systems are not yet commercially viable because the operating temperature for yeast of 28° C. is too low for the 50° C. conditions required by cellulase; operation at a compromise temperature of 37° C. is inefficient and prone to microbial contamination.
A third approach to overcoming the shortage of β-glucosidase is to use genetic engineering to overexpress the enzyme and increase its production by Trichoderma. This approach was taken by Barnett, Berka, and Fowler, in “Cloning and Amplification of the Gene Encoding an Extracellular B-glucosidase from Trichoderma reesei: Evidence for Improved Rates of Saccharification of Cellulosic Substrates,” BioTechnology, Volume 9, June 1991, p. 562-567, herein referred to as “Barrett, et al.”; and Fowler, Barnett, and Shoemaker in WO 92/10581, “Improved Saccharification of Cellulose by Cloning and Amplification of the B-glucosidase gene of Trichoderma reesei,” herein referred to as Fowler, et al.”
Both Barnett, et al. and Fowler, et al. describe the insertion of multiple copies of the β-glucosidase gene into Trichoderma reesei strain P40. Both groups constructed plasmid pSASβ-glu, a transformation vector containing the genomic T. reesei β-glucosidase gene and the amdS selectable marker. The amdS gene is from Aspergillus nidulans and codes for the enzyme acetamidase, which allows transformed cells to grow on acetamide as a sole source of nitrogen. T. reesei does not contain a functional equivalent to the amdS gene and is therefore unable to utilize acetamide as a nitrogen source. The transformed cells contained 10 to 15 copies of the β-glucosidase gene and produced 5.5-fold more β-glucosidase than the untransformed cells.
The enhanced production of β-glucosidase obtained by Barnett, et al. and Fowler, et al. is not sufficient to alleviate the shortage of β-glucosidase for cellulose hydrolysis. The amount of β-glucosidase made by natural Trichoderma strains, for example, must be increased at least 10-fold to meet the requirements of cellulose hydrolysis.
When overexpressing proteins in Trichoderma, one strategy is to link the gene of interest directly to the cbh1 promoter or to the cbh1 secretion signal. Since CBH1 is the most abundant protein produced by Trichoderma under cellulase-inducing conditions, the cbh1 promoter and secretion signal are thought to be very effective in directing the transcription and secretion of proteins encoded by a gene positioned after them in a genetic construct. Such a strategy has been successfully used to overexpress proteins from Trichoderma and other microorganisms (Margolles-Clark, Hayes, Harman and Penttila, 1996, “Improved Production of Trichoderma harzianum endochitinase by expression in Trichoderma reesei”, Appl. Environ. Microbiol. 62(6): 2145-2151; Joutsjouki, Torkkeli and Nevalainen, 1993, “Transformation of Trichoderma reesei with the Hormoconis resinae glucoamylase P (gamP) gene: production of a heterologous glucoamylase by Trichoderma reesei”, Curr. Genet. 24: 223-228; Karhunen, Mantyla, Nevalainen and Suominen, 1993, “High frequency one-step gene replacement in Trichoderma reesei 1. Endoglucanase I overproduction”, Mol. Gen. Genet. 241: 515-522).
Another example of an industrial enzyme produced within fungal expression systems includes xylanase. Some of the most important commercial xylanases are classified as Family 11 xylanases. A xylanase enzyme is classified in Family 11 if it possesses the amino acids common to Family 11, including two glutamic acid residues serving as the essential catalytic residues. These residues are amino acids 86 and 177 by Trichoderma reesei xylanase II numbering. The amino acids common to Family 11 xylanases are described in Wakarchuck, et al, Protein Science 3:467-475 (1994).
The expression of pharmaceutically important heterologous proteins, for example insulin (Goeddel D. V. et al., 1979, Proc. Nat. Acad. Sci. 76 106-110), and blood coagulation factor Xa (Smith D. B. 1988, Gene 67: 31-40), have been reported using bacterial expression systems. Similarly, U.S. Pat. No. 4,751,180 discloses the expression of a heterologous protein in yeast, including insulin and IgF-2.
The heterologous production of bovine prochymosin has been reported using the filamentous fungi Trichoderma reesei (Harkki A. et al., 1989, Bio/Technol. 7:596-603), with the genetic construct comprising a cbh1 (cellobiohydrolase I gene) regulatory region and terminator, either the cbh1 or the chymosin signal sequence, and optionally an intervening region obtained from cbh1. Margolles-Clark E. et al., (1996, App Environ Microbiol., 62:2152-2155) disclose the expression of T. haraianum endochitinase using cbh1 promoter from T. reesei. Similarly, proteins of interest, for example chymosin, have also been produced in the filamentous fungi Aspergillus nidulans and A. awamori, using genetic constructs comprising the regulatory region and secretion signal from the glaA (glucoamylase) gene, a signal sequence from either glaA or chymosin, and an intervening region from glucoamylase (EP 215,594). In both of these latter cases, transcription of the protein product within the host was not a limiting factor of heterologous protein production. However, secretion of the protein product out of the host was low and resulted in poor extracellular protein recovery.
In an attempt to increase the extracellular accumulation of heterologous protein production within a filamentous fungi expression system, Lawlis (1997, U.S. Pat. No. 5,679,543) disclosed the use of a multi-component fusion polypeptide to enhance secretion and extracellular accumulation of a protein of interest. The genetic constructs were complex encoding a fusion protein comprising four parts and included a signal peptide, a secreted polypeptide or portion thereof (a carrier protein), a clevable linker polypeptide, and the desired polypeptide for which expression is desired. Increased levels of protein secretion were attributed to the use of a glaA signal sequence fused to full length glucoamlyase (the carrier protein), or other protein that is secreted within the host, which was then fused to the cleavable linker and protein of interest (chymosin). Such construct were found to increase the secretion of the fusion polypeptide when expressed in the filamentous fungi A. nidulans. 
Even though increased secretion was noted within U.S. Pat. No. 5,679,543, using these four-component fusion proteins, the production of the expression vectors is complex. This expression system requires the use of a six-part genetic construct that expresses a complex four-part protein product with an expressed and variable carrier protein. Furthermore, approximately 50% of the desired expressed product is not recovered as it comprises the carrier protein, and this increases costs associated with post expression handling and purification of the desired protein. Significant post-secretion processing including linker cleavage and acidification of the medium is also required for recovery of the desired final protein product.
There is required within the art a simplified expression system that results in high levels of expression and secretion of a protein of interest from a host cell. Preferably, the genetic constructs used within this expression system comprise few component parts, so that the chimeric construct is easy to prepare. Furthermore, the levels of expression and secretion using such genetic construct should be high, and preferably, little or no downstream manipulations are required for harvesting the protein of interest.
It is an object of the invention to overcome disadvantages of the prior art.
The above object is met by the combinations of features of the main claims, the sub-claims disclose further advantageous embodiments of the invention